Cells of diverse organisms, from cyanobacteria to humans, execute temporal programs of physiological processes that are driven by endogenous timing mechanisms. The overall goal of this project is to understand the biochemical events that allow a cell to keep track of time, execute activities according to a temporal program, and synchronize the internal clock with the external solar cycle. These processes are crucial to wellness, and dysfunction results in disease. The specific aims address the mechanisms of each of these aspects of biological timing in the cyanobacterium Synechococcus elongatus, the simplest model organism known to have a 24-h biological (circadian) clock. The components of the circadian oscillator are known (proteins KaiA, KaiB, and KaiC), their structures have been solved, and the circadian rhythm in phosphorylation of KaiC can be reconstituted in vitro. The organism is amenable to genetic manipulation and its circadian rhythms of gene expression can be visualized through luciferase reporter genes. Clock mechanism across all groups of organisms is operationally divided into the oscillator, the input pathways that synchronize the oscillator with the solar day and provide internal coordination among distinct molecular oscillators, and the output pathways that enable the oscillator to control cellular functions. This project will provide insight into the operation of each of these aspects of the clock, their feedbacks and functional overlaps, and their integration with other activities of the cell. The specific aims will: (1) Define the physical, biochemical, and redox interactions of input pathway components, (2) Assess the modulation of oscillator activity by input and output components, and (3) Define the biochemical states of KaiC that stimulate the major output pathway. Mass spectrometry will be used to determine the identity and activity of quinone redox cofactors that are bound by the oscillator, and to define the physical interactions among input, output, and oscillator proteins, as well as other cellular components. Genetic and biochemical methods will identify the factor responsible for a newly discovered KaiC-stimulating activity and the signaling partner of the input factor CikA. Oscillators reconstituted in vitro will be used to test the direct influence on cyclic KaiC phosphorylation of input and output components, and of redox modulation. Both biochemical assays and a genetic reporting system will be used to determine the biochemical state of the oscillator that triggers the downstream signaling system. Insights into a circadian oscillator and its integration with cellular metabolism will inform medical advances, such as understanding the circadian link to metabolic syndrome and improving the efficacy of pharmaceutical interventions.